Understanding mechanisms of RNA catalysis remains an intriguing challenge, one that has grown in significance since the recent demonstration that the ribosome is a ribozyme. Several ribozymes catalyze the same reversible RNA cleavage reaction but adopt different structures and exploit distinct kinetic and catalytic mechanisms. Comparison of these mechanisms provides insight into the diversity of catalytic strategies that are available to RNA enzymes. The unique pH and metal ion dependencies of hairpin ribozyme catalysis was the first sign that ribozymes can accomplish catalysis exclusively through the use of RNA functional groups with no requirement for direct coordination of metal cations to phosphate, ribose or water oxygens. Until now, the difficulty of distinguishing structural from catalytic effects of experimental manipulations seriously limited studies of the hairpin catalytic mechanism. Two recent developments have transformed the field in this regard. First, a crystal structure has been solved for a hairpin ribozyme complex with a substrate analog that provides the first high-resolution view of the functional groups that comprise the active site. Second, hairpin ribozyme variants have been developed that allow catalytically important nucleotides to be modified without disrupting the functional structure. The proposed research will develop a detailed model for the hairpin ribozyme catalytic mechanism by elucidating the roles of specific RNA functional groups in catalytic chemistry and active site architecture. The principal investigator will test whether ribozyme functional groups mediate general acid base catalysis by examining pH-rate profiles for reactions of ribozyme variants missing specific active site nucleobases or with active site nucleobases replaced by nucleobase analogs that differ in acid or base strength. Preliminary data suggest that cationic nucleobases might provide electrostatic stabilization to negative charge developing in the transition state or stabilize oxyanion nucleophiles or leaving groups. Dr. Fedor e will examine this notion using an abasic ribozyme rescue strategy and through covalent incorporation of nucleobase analogs that differ in the propensity to undergo ionization. Finally, she will examine how structural and biochemical features of the active site influence catalysis through positioning and orientation of reactive groups.